Protein synthesis in higher eukaryotes is highly regulated and is determined primarily by the phosphorylation state of the alpha subunit of the eukaryotic initiation factor 2 (eIF-2). Phosphorylation of this subunit by several specific inhibitory kinases inactivates eIF-2 thereby inhibiting protein synthesis. Alternatively, there are proteins within this regulatory pathway that interact with eIF-2 to promote protein synthesis.
Methionine aminopeptidase 2 (also known as MetAP2 and eIF-2 associated protein, p67) is a cellular glycoprotein that promotes protein synthesis in the presence of active eIF-2 kinases by protecting the eIF-2 alpha subunit from phosphorylation (Li and Chang, Biochem. Biophys. Res. Commun., 1996, 227, 152-159; Li and Chang, Biochim. Biophys. Acta., 1995, 1260, 333-336). Methionine aminopeptidase 2 protects eIF-2 by binding to the gamma subunit of the three-subunit eIF-2 complex (Ray et al., Biochemistry, 1993, 32, 5151-5159). Disclosed in U.S. Pat. Nos. 5,885,820 and 5,888,796 respectively are the nucleotide and polypeptide sequences of human methionine aminopeptidase 2 (Chang, 1999; Chang, 1999).
The methionine aminopeptidase 2 protein is an N-terminal processing enzyme which removes the N-terminal amino acid, methionine, from nascent proteins. This enzymatic activity requires cobalt and affects the stability, function and localization of the target protein (Arfin et al., Proc. Natl. Acad. Sci. U.S.A., 1995, 92, 7714-7718). Antibodies to methionine aminopeptidase 2 have been shown to inhibit protein synthesis (Ray et al., Biochemistry, 1993, 32, 5151-5159).
Expression of methionine aminopeptidase 2 is highly regulated through the process of deglycosylation and degradation (Chakraborty et al., Biochemistry, 1994, 33, 6700-6706; Datta et al., J. Biol. Chem., 1989, 264, 20620-20624) and, in mammals, methionine aminopeptidase 2 has been shown to be activated during serum starvation (Gupta et al., J. Biol. Chem., 1997, 272, 12699-12704), heat shock (Chatterjee et al., Biochem. Biophys. Res. Commun., 1998, 249, 113-117) and viral infection (Bose et al., Arch. Biochem. Biophys., 1997, 342, 362-372; Saha et al., Arch. Biochem. Biophys., 1997, 342, 373-382). The deglycosylation and subsequent degradation of methionine aminopeptidase 2 has been correlated with increased phosphorylation of eIF-2 in osteosarcoma cells at certain stages of the cell cycle (Datta et al., Exp. Cell. Res., 1999, 250, 223-230). The pharmacological modulation of methionine aminopeptidase 2 activity or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, inhibitors reported in the art that target methionine aminopeptidase 2 reduce the activity of the enzyme and are being tested as therapeutic agents. These agents, known as ovalicin and fumagillin, are fungal metabolites that inhibit angiogenesis and endothelial growth (Griffith et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 15183-15188). Derivatives of fumagillin have been synthesized and shown to have immunosuppressive properties as well (Griffith et al., Chem. Biol., 1997, 4, 461-471; Liu et al., 1998; Turk et al., Bioorg. Med. Chem., 1998, 6, 1163-1169).
Other strategies aimed at modulating methionine aminopeptidase 2 function have involved the use of antisense expression vectors. Using an antisense construct in reverse orientation containing the methionine aminopeptidase 2 gene, Gupta et al. demonstrated that methionine aminopeptidase 2 is necessary for protein synthesis in KRC-7 rat hepatoma cells (Gupta et al., Gene Expr., 1995, 5, 113-122). It has also been reported that lowering the levels of methionine aminopeptidase 2 using the same antisense construct leads to the induction of apoptosis, or programmed cell death, in these cells suggesting that a certain level of methionine aminopeptidase 2 is required for normal cell growth (Datta and Datta, Exp. Cell. Res., 1999, 246, 376-383).
There remains, however, a long felt need for additional agents capable of effectively inhibiting methionine aminopeptidase 2 function and antisense technology is emerging as an effective means for reducing the expression of specific gene products. This technology may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of methionine aminopeptidase 2 expression.
The present invention provides compositions and methods for modulating methionine aminopeptidase 2 expression.